In this work the optical fiber glucose and lactatebiosensors were developed by using fluorescent dye and enzymeimmobilized on the end tip of an optical fiber. 3-Glycidyloxypropyl)methyldiethoxysilane (GPTMS), (3-Aminopropyl)trimethoxysilane (APTMS) and Methyltrimethoxysilane(MTMS) were used to immobilize glucose oxidase (GOD),lactate oxidase (LOD) and ruthenium(II) complex (tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II), Ru(dpp)32+) asoxygen sensitive fluorescent dye. MTMS sol-gel was an excellentsupporting material for the immobilization of Ru(dpp)32+,GOD, and LOD on the optical fiber. Storage stability of theoptical fiber glucose sensor was kept constant over 20 days,while the optical fiber lactate sensor had constant storage stabilityover 17 days. The optical fiber glucose and lactate biosensorsalso maintained good operational stability for 20 hoursand 14 hours, respectively. The activities of the immobilizedenzymes were most excellent at pH 7 and at 25oC. On-line monitoringof glucose and lactate in a simulated process was performedwith the optical fiber glucose and lactate biosensors. On-line monitoring results were agreed with those of off-linedata measured with high performance liquid chromatography(HPLC).